11-hydroxy-3, 17-dioxo-4, 6-androstadiene and its preparation



United States Patent 11-HYDROXY-3,17-DIOXO-4,6-ANDROSTADIENE AND ITS PREPARATION Jack W. Ralls, Morton Grove, 111., assignor to G. D. Searle & Co., Chicago, 111., a corporation of Illinois No Drawing. Application July 5, 1952, Serial No. 297,405

1 Claim. c1. 260--397.45)

The present invention relates to a new group of cyclopentanophenanthrene compounds and more particularly to hydroxy-3-oxo-4,6-androstadienes and methods for their production. I have found that upon oxidation by adrenal tissues of a 3-oxo-4,6-androstadiene there can be made to occur two competitive processes which can be represented structurally as follows:

In these structural formulas =R can represent such oxygen-containing radicals as =0,

acetyl hydroxyacetyl and 2,715,640 Patented Aug. 16, 1955 is dehydrobrominated with pyridine to yield the 3-oxo- 4,6-androstadiene.

B. A 5-androsten-3-ol is oxidized by quinone in the presence of aluminum isopropoxide in toluene.

C. A 3-oxo-4-androstene is brominated with N-bromosuccinimide to form the 3-oxo-6-bromo-4-androstene which is then dehydrobrominated by use of collidine to yield the 3-oxo-4,6-androstadiene.

While the 3-oxo-4,6-androstadienes are not known to exist in nature, the literature lists the preparation of a number which are suitable for the hydroxylation reaction claimed. The compounds which I find to be valuable starting materials are 3,17-dioxo-4,6-androstadiene, 3-oxo-17-hydroxy-4,6-androstadiene, 3-oxo-17-acetyl-4,6- androstadiene, 3 oxo-l7- (jS-hydroxyacetyl) -4,6androstadiene.

It has been my experience that, while some oxidation may be achieved by use of adrenal homogenates and mashes, the oxidation can be carried out in optimal yield by exposing the steroid to circulation through a surviving mammalian adrenal gland using a perfusing medium such as blood.

The process is probably of an aerobic nature. However, it has been found that a moderately aerated perfusion medium gives satisfactory yields. A typical procedure is described below. It will be obvious to biochemists skilled in the art that numerous modifications of this procedure may be practiced and the invention is not to be construed as limited by the experimental details set forth.

1.0 part of the 3-oxo-4,6-androstadiene is stirred with 5000 parts of citrated beef blood and 5000 parts of 0.86% aqueous sodium chloride solution. This solution is perfused three times through a surviving beef adrenal, cannulated through the vein and having a finely lacerated surface. The blood is then extracted exhaustively with isopropyl acetate. Evaporation of the solvent in vacuo yields a mixture of the 11-hydroxy-3-oxo-4,6-androstadiene and the 2-hydroxy-3-oxo-4,6-androstadiene and some unreacted starting material. By this method there are obtained 3,17-dioxo-4,6-androstadien-1l-ol, 3-oxo- 4,6-androstadiene-11,17-diol, 3-oxo-17-acetyl-4,6-androstadien-l l-ol, and 3-oxo-17-(fi-hydroxyacetyl)-4,6-androstadien-l l-ol from the starting materials listed above. Under these conditions the corresponding 2-01 derivatives are usually obtained in relatively smaller yield. For some medicinal purposes, it is most advantageous to use the mixture of hydroxylated products obtained by evaporation of the isopropyl acetate since there may be some synergistic efiect between the two. Where a separation of the hydroxy compounds is desirable, this can be conveniently accomplished by chromatographic absorption on silica gel and selective elution by a mixture of benzene and ethyl acetate.

By a preliminary rinsing with benzene, minor amounts of cholesterol and gland constituents are removed. This removal is carried further by use of small amounts of a 9:1 mixture of benzene and ethyl acetate. On further washings with 9:1 to 4:1 mixtures, the unreacted starting material is removed. The next fraction to be eluted as the relative concentration of the ethyl acetate is raised is the ll-hydroxy compound, the Z-hydroxy compound being obtained last.

The two types of hydroxy compounds are distinguishable in certain important respects. The 2-hydroxy compound, being an a-ketol, gives a positive blue tetrazoliurn test, the ll-hydroxy compound does not. Further, the 2- hydroxy compound is readily esterified by such agents as acetyl chloride and acetic anhydride. The ll-hydroxy group, being sterically hindered is difiicult to acylate; actually there is reason to believe that the process described hereinabove favors hydroxylation in the 11-5 po- 'of benzene.

3 sition, a position in which the hydroxyl group cannot be acylated under normal conditions.

A typical example for this separation follows: 1.37 parts ofresidue obtained upon evaporation of the isopropyI acetate from an extract resulting from the perfusion of 3,17rdioxo-4,6-androstadiene are dissolved in a solution of 20 parts of isopropyl acetate and 480 parts chromatography column containing 137 parts of silica gel. A small quantity of byproducts is eluted by pouring 900 parts of benzene and 900 parts of a 9:1 mixture of ethyl acetate and benzene through the column. The column is next eluted with two successive 450-part portions of a 9:1 mixture of benzene and ethyl acetate and two 450-part portions of a 4:1 mixture of benzene and ethyl acetate. The resulting four extracts, upon evaporati'on, yield unreacted 4,6-androstadiene-3,17-dione. The column is next eluted with two 450-part portions of a 7 2:1 mixture of benzene and ethyl acetate and then with It gives a positive Zimmermann reaction and a negative blue tetrazolium test.

The column is next eluted with three 450-part portions of a 1:1 mixture of benzene and ethyl acetate. The second and third of these fractions are concentrated and the resulting crystallized from ethyl acetate. It melts at about The resulting solution is. poured onto a' 2-hydr0xy-4,6-androstadiene-3,17-dione 2112l4 C. This compound shows a specific rotation a- =+139 in an 0.55% chloroform solution and has an ultraviolet maximum at 283 millimicrons with an extinction coeflicient of 26,100. mula 0 CH3 7 I It gives a positive Zimmermann reaction as well as a positive blue tetrazolium test confirming the presence of a ketol structure.

In this instance the ratio of Z-hydroxy compound is about 5:3.

I claim: A compound of the formula References Cited in the file of this patent UNITED STATES PATENTS 2,166,877 Reichstein July 18, 1939 2,183,589 Reichstein Dec. 19, 1939 2,236,574 Koester Apr. 1, 1941 2,332,815 Ruzicka Oct. 26, 1943 2,403,683 Reichstein July 9, 1946 2,602,769 Murray et al July 8, 1952 2,691,030 Murray Oct. 5, 1954 OTHER REFERENCES Fieser et al.: National Products Related to Phenanthrene, 3rd ed., pp. 409-410 (1949).

It has the structural for-' the yields of ll' h ydi'oxy to 

